105 results about "Lysobacter enzymogenes" patented technology

The invention provides a biocontrol bacterial strain for preventing and treating plant diseases and its bacterial agent, belonging to the field of biological control. The strains used are Gram-negative bacteria, identified as Lysobacter enzymogenes, and the strain code is OH11. Strain OH11 has no flagella but has slippage, can produce various extracellular hydrolytic enzymes including chitinase, β-1,3-glucanase and protease, and can effectively inhibit the growth of fungi and bacteria. The antibacterial zone diameters of strain OH11 against Sclerotinia sclerotiorum and Phytophthora capsici were both greater than 22.0 mm; the antagonism against potato ring rot was stronger, and the diameter of the inhibition zone reached 50 mm. The OH11 strain was inoculated into the seed tank, cultivated to the logarithmic growth phase, and the seed liquid was connected to the production tank for cultivation. The medium used in the production tank was the same as that of the seed tank. The liquid fermentation adopts aerobic submerged fermentation and fed-batch process, the dissolved oxygen is 15%-20%, the fermentation temperature is 30°C, the fermentation time is 72h, and the initial pH value is 7.5. After the fermentation is completed, the culture solution is taken out of the tank and directly packed into liquid dosage forms with plastic packaging barrels or packaging bottles, or subpackaged into solid dosage forms with peat adsorption packaging bags. The biocontrol strain OH11 can effectively control plant pathogenic fungi, bacteria, nematodes and other diseases, and the overall control effect is 50%-70%. In the greenhouse pot experiment, the control effects of OH11 on pepper blight and tomato bacterial wilt reached 83.6% and 86.4%, respectively. Strain OH11 has the characteristics of broad antibacterial spectrum, high activity, and environmental safety. In today's serious pesticide pollution, zymolysobacterium and its bacterial agent will be a good substitute.

The invention discloses a method for synthetizing secretion lysozyme by middle silkgland cell of silkworm. The method comprises the following steps: building a pBSer1hLYZ (lysozyme)-A3EGFP (Enhanced Green Fluorescent Protein) plasmid for synthetizing secretion lysozyme by the silkworm; then, introducing the plasmid and an assistant plasmid capable of providing transposase into a silkworm germ cell according to the microinjection transgenosis silkworm technology; according to the transposition characteristics of a piggyBac transposon, introducing a green fluorescent protein gene and a lysozymegene into a silkworm gene group, to obtain stable heredity and expression so as to create the transgenosis silkworm capable of synthetizing secretion lysozyme by middle silkgland cell of silkworm by specificity; further, hybridizing the transgenosis silkworm with sericin silkworm; carrying out back crossing on the hybridized descendant with the sericin silkworm for 3-5 generations; and finally, carrying out selfing on the obtained product to carry out homozygosis on the lysozyme gene so as to obtain the new species of the transgenosis silkworm of the secretion lysozyme. According to the method, a basis for improving lysozyme production efficiency and lowering production cost is laid.

The invention discloses a housefly cecropin-human lysozyme fusion protein, and a preparation method and application thereof. A sequence of the housefly cecropin-human lysozyme fusion protein of the invention is shown by SEQ ID No.1. The housefly cecropin-human lysozyme fusion protein comprises a mature peptide sequence of housefly cecropin, a polypeptide linker sequence and the mature peptide sequence of human lysozyme. The housefly cecropin-human lysozyme fusion protein is prepared by the following steps of: according to the gene sequences of the housefly cecropin and the human lysozyme, designing a synthetic primer with esherichia coli preferred codons; performing PCR amplification by using a splicing overlap extension method to obtain the gene sequence of the fusion protein so as to construct recombinant prokaryotic expression plasmids; transferring the recombinant prokaryotic expression plasmids into host cells to obtain engineering bacteria expressing the fusion protein; and performing fermentation culture, separation and purification to obtain the housefly cecropin-human lysozyme fusion protein of the invention. The fusion protein of the invention has the characteristics of the housefly cecropin and the human lysozyme per se, has relatively more remarkable antibacterial activity and antibiotic activity, can be used for preparing antibacterials or bacteriostatic medicaments, and has wide application prospect.

The invention provides an optimized high-activity human lysozyme gene as well as expression vectors thereof. The gene sequence is optimized according to the codon bias of Pichia pastoris and the human lysozyme gene is artificially synthesized; recombinant plasmids pUC18-T-HZ and pPICZ(alpha)A-HZ are constructed; the recombinant plasmid pPICZ(alpha)A-HZ is linearized and then transfected to Pichiapastoris, and positive transformants are obtained by screening. The study results show that the recombinant Pichia pastoris can secrete and express high-activity human lysozyme, and the expression product has significant bacteriostatic activity. According to the invention, the Pichia pastoris expression system is adopted to produce the human lysozyme with high expression level and high activity, so as to overcome the problems in the prior art, such as high cost, low expression level and low activity. The production and the promotion and application of the high-activity and low-cost lysozyme can bring considerable economic and social benefits for the development of feed and animal husbandry industries in China and huge ecological benefit as well.

The invention discloses a method for producing active antimicrobial substance HSAF by optimizing lysobacter enzymogenes OH11 with response surface method. The method comprises: strain activation, seed liquid culture, fermentation culture, and extraction and detection of HSAF in fermentation broth. A fermentation medium comprises following raw materials according to a ratio: 8.00g/L of soy flour, 7.89g/L of glucose and 0.72g/L of CaCl2. The liquid containing amount, the culture temperature and the fermentation period are 22%, 26 DEG C and 58h respectively. Inert substances are added into HSAF extraction process to eliminate an emulsion problem, so that the fermentation liquid HSAF can be accurately quantitative, and ultimately the yield of HSAF can be up to 440.26mg/L, which is 15 times that produced under conventional conditions (10% TSB, 29.24mg/L). The method has the advantages of being extensive in raw materials, low in cost, capable of effectively reducing the production cost of HSAF, and providing important technical parameters for the future production of HSAF.

The invention particularly relates to Lysobacter enzymogenes 1-T-1-4 and application thereof. The Lysobacter enzymogenes 1-T-1-4 can be prepared into aquae or wettable powder and is used for preventing and treating diseases such as panax notoginseng root rot and amorphophallus konjac soft rot through acting on panax notoginseng or amorphophallus konjac.

The invention belongs to the field of bio-genetic engineering and provides a DNA segment for efficient expression of egg white lysozyme by employing recombinant pichia pastoris and an expression method thereof. The method comprises the following steps: directly obtaining a gene of egg white lysozyme by gene synthesis means; carrying out operations such as digestion and connection, and then introducing the gene into an eukaryotic expression vector pPIC9K; transforming into pichia pastoris GS115, screening high-copy recons, and then realizing efficient expression of the recombinant egg white lysozyme in vitro through methanol induction. The method is not limited by a raw material source; the copy number of the lysozyme can be increased; the expression amount and the activity of the enzyme are improved; the later separation and purification technologies are simplified; large-scale industrialized production of the egg white lysozyme is realized; and the DNA segment has the characteristics of being stable, pure, and high in biological activity, and can be widely applied to many fields such as pharmacy and feeds.

The invention relates to a microorganism from the species Lysobacter enzymogenes, having nematicidal activity and the ability to promote plant growth. The invention also provides methods for obtaining a biomass of said microorganism, as well as methods for biologically controlling nematodes, for treating and preventing plant infection caused by nematodes, and for promoting plant growth based on the use of said microorganism or of phytosanitary products obtained therefrom.

The invention relates to a preparation method of a natural colorless biological mildew-proof anticorrosive bactericide. The preparation method comprises the following steps: firstly inoculating lysobacter enzymogenes C3 and a circle of seeds in a conical flask, and culturing on a shaking table to obtain a lysobacter enzymogenes C3 seed liquid; putting a liquid culture medium in a fermentation tank and then inoculating the lysobacter enzymogenes C3 seed liquid for culture to obtain a high-activity lysobacter enzymogenes C3 mildew-proof anticorrosive bactericide; heating the high-activity lysobacter enzymogenes C3 mildew-proof anticorrosive bactericide for 2-10 minutes at the temperature of 60 to 100 DEG C in order to perform thermal treatment; finally adding modified active carbon into the bactericide to perform a decoloration reaction to obtain the natural colorless biological mildew-proof anticorrosive bactericide, wherein the amount of the added modified active carbon is 0.5-3.0 percent of the weight of the high-activity lysobacter enzymogenes C3 mildew-proof anticorrosive bactericide which is in light brown. Due to acidic modification, an acidic oxygen-containing functional group on the surface of the active carbon is enabled to have polarity property, so the adsorption on compounds which are relatively high in polarity is enhanced, the adsorption capacity of the active carbon is improved, and the preferential adsorption of the active carbon on specific impurities like pigments is improved.

The invention relates to a novel member LYC2 of lysozyme gene family. The invention provides the cDNA sequence encoding for the novel lysozyme, the polypeptide encoded by the sequence, as well as the method for producing said novel human lysozyme utilizing recombinant technology. The invention also provides the use of the novel human lysozyme.

The invention discloses a lysozyme mutant with improved specific activity, belonging to the technical field of bioengineering. The invention discloses the human lysozyme mutant which is obtained by adding hydrophobic oligopeptide Val-Ile-Pro-Leu-Phe to the C end of human lysozyme through genetic engineering. The specific activity of the human lysozyme mutant is improved by 126%. The invention alsodiscloses a recombinant plasmid containing a mutant human lysozyme gene, and a recombinant genetically-engineered pichia pastoris strain which is obtained by transforming pichia pastoris with the recombinant plasmid and capable of efficiently expressing the human lysozyme mutant. The obtained human lysozyme mutant has the characteristics of high specific activity and simple preparation, has potential clinical application value, and also has wide application in feed and food industries.

The invention provides egg-white lysozyme and a preparation method thereof. The coding sequence of polypeptide amino acid with protein activity of the egg-white lysozyme is a sequence show in a sequence table 400<1>. The preparation method comprises the following steps: (1) obtaining genes of the egg-white lysozyme; (2) constructing recombinant plasmids; (3) constructing genetic engineering strains; and (4) carrying out producing method of genetic engineering recombinant protein. The egg-white lysozyme has the characteristics of stability, purity and high bioactivity and can be widely applied multiple field, such as pharmacy and feed. According to the preparation method, the genetic engineering strains act on fermentatively synthesized lysozyme, by adopting constructed engineering strains, the copy number of enzyme genes can be greatly increased, the expression quantity of the enzyme is improved, and thus the efficient synthesis of the egg-white lysozyme is achieved and the preparation method of the egg-white lysozyme is not limited by the source of raw materials.

The invention discloses an Escherichia coli expression vector capable of controlling the self-cracking of a host bacterium, which belongs to the technical field of microbes and gene engineering. In the invention, the composition of the codon of a lysozyme gene in a T4 phage and a restriction endonuclease identification site are optimized, and the optimized T4 lysozyme gene mutation lyMu and a modified Escherichia coli expression vector are recombined to form a recombinant expression vector pEly. The expression vector pEly can be used for inducing expression of an exogenous gene, and when the exogenous gene is induced to express, T4 lysozyme is expressed. The cell undergoing induction expression is treated by ethylene diamine tetraacetic acid (EDTA) solution and then self-cracks, so that the product of expression in the cell is released. The expression vector obtained by implementing the invention can realize the express of various exogenous proteins in Escherichia coli hosts, the host bacteria release the products of expression after undergoing EDTA treatment, and thus, the recovery of the products of the expression can be promoted. The expression vector has an application prospect in fields of enzymic preparation production, polypeptide medicine production and the like.

The invention relates to a method for screening of chlamys farreri G-type muramidase gene polymorphism markers and the assistant breeding method thereof, belonging to shellfish molecular marker assistant breeding technology in the filed of aquatic organism technology, mainly comprising the steps of: cloning partial sequence of promoter region of a chlamys farreri G-type muramidase gene, preparing disease-resistance population and sensitive population, screening of disease-resistance G-type muramidase gene markers, quickly screening the disease-resistance population and building gene marker assistant breeding technology. According to the invention, promoter region sequence of the chlamys farreri G-type muramidase gene is cloned, and its polymorphism sites are screened. The occurrence frequency of -391 AG individual in disease-resistance population is obviously higher than that in sensitive population, therefore, resistance-related gene marker assistance breeding method is built with -391 AG as chlamys farreri resistance-related G-type muramidase gene marker. The invention has the characteristics of strong pertinence, high breeding efficiency, simple and quick operation, etc., and is suitable for screening of shellfish resistance-related markers and breeding of disease-resistance fine varieties.

The invention discloses a procambrus clarkii type i lysozyme gene of which nucleotide sequence is shown in the SEQ ID NO. 1 and provides a recombinant expression and purification method thereof. The invention also provides a lysozyme polypeptide coded by the procambrus clarkii type i lysozyme gene and applications of the gene sequence and the lysozyme, wherein the amino acid sequence of the lysozyme polypeptide is shown in SEQ ID NO. 2.; and the lysozyme of the invention has inhibitory activity to both Gram-positive bacteria and Gram-negative bacteria. The lysozyme gene of the invention can be transferred in crops through the gene engineering method to obtain crops with antibacterial ability. The recombinant lysozyme obtained in the invention can be used in antibacterial feed additives and the fields of food preservation, gene transformation of animals and plants and drug development.

The invention relates to lysozyme, specifically to a Mytilus edulis G-type lysozyme gene and a recombinant protein and application thereof. The G-type lysozyme gene has a base sequence as represented by SEQ ID No. 1 in a sequence table. Through PCR technology, a gene fragment coding the mature peptide of the G-type lysozyme gene is amplified and cloned into a pET21(a) expression vector, and prokaryotic in-vitro recombinant expression in escherichia coli BL21(DE3)plysS is realized. After Ni-affinity chromatographic purification and dialysis renaturation, a recombination product has inhibitory effects on a variety of Gram positive and negative pathogens and especially has a substantial effect on pseudomonas putida, and minimal inhibitory concentration is 2.39 to 4.79 mu mol/L. The invention provides bases for further research on immune defense mechanism of economic shellfish and lays a foundation for disease control, genetic breeding and feed additives for aquatic livestock.

The invention discloses a method for improving pig lysozyme by fusing an N-terminal with an HLH functional domain, and belongs to the field of bioengineering. The method comprises the steps of design of a fused pig lysozyme gene and expression and renaturation of an escherichia coli expression system. By means of detection of antibacterial activity, the fused pig lysozyme has obvious resistance to gram-positive bacteria and can kill various gram-positive bacteria simultaneously, and an excellent case is provided for improving the antibacterial activity of the lysozyme. The fused pig lysozyme preparation technology has the advantages of being simple and efficient, the product can reach electrophoretic purity, and the purity is 90% or above.

The present invention provides production process of human lysozyme as AIDS treating medicine with plant as bioreactor. Human lysozyme gene is cloned from human placenta and constructed into prokaryotic expression vector for efficient expression. Plant expression vector is constructed in lettuce, and the human lysozyme gene is introduced into lettuce nuclear genome to realize the efficient expression of human lysozyme gene in higher plant and to obtain one kind of lettuce containing human lysozyme. Human lysozyme mass produced in the said method may be used in treating AIDS, tumor and viral diseases.

The invention relates to molecular biology, in particular to a meretrix lysozyme gene and a coding protein and application thereof. The meretrix lysozyme gene is whole cDNA series 552bp of the meretrix lysozyme which is amplified in the meretrix by using a cDNA library and an RACE technique, wherein a whole reading frame is 441bp; a 5' non-coding region is 15bp; a 3' non-coding region is 96bp; the whole coding protein contains 146 amino acids; 1 to 15 of a coding sequence is a signal peptide sequence; and a mature peptide contains 131 amino acids including 14 cysteine. An expression primer is designed according to the obtained sequence, and after the overall length of the expression primer is amplified, the expression primer is subjected to enzyme cutting to connect into an expression vector pGEX-4T-1; and then a recombinant plasmid is converted into Escherichia coli BL21, and subjected to IPTG inducing, GSTrap FF affinity column purifying and thrombin enzyme cutting GST to form recombinant lysozyme. The lysozyme has wide medicinal and industrial value and wide application prospect, and can be used as a bactericide, an immune potentiator of shellfish growth, an additive of medicaments, feeds, foods and the like, and the like.

The invention discloses a gene chip for detecting transgenic cattle and a preparation method and application thereof. The chip provided by the invention contains solid phase carrier and an oligonucleotide probe fixed on the carrier, wherein the oligonucleotide probe contains a detection probe and a quality control probe; and the detection probe is a specific probe of mitochondrial genes of cattle, sheep and pig, human lactoferrin gene, human lysozyme gene, human-alpha-lactoalbumin gene, green fluorescent protein gene and neomycin phosphotransferase gene. By adopting the gene chip and the construction method to detect transgenic cattle, the accuracy, specificity and sensitivity are all higher; and the gene chip is suitable for the high throughout screening of transgenic cattle.

The invention discloses a targeted exogenous gene integration method. Taking human lysozyme as an example, a human lysozyme gene is targetedly integrated to a bovine as1-casein loca by a gene targeting method so that a lysozyme gene captures a complete as1-casein regulatory sequence and the regulatory sequence on the casein loca; therefore, the overexpression of the human lysozyme in a bovine mammary gland is realized. By using the new method, a target gene can be targetedly integrated to a loca which contributes to self-expression; various shortcomings in conventional exogenous gene random integration in transgene are overcome; and the success rate of transgenic breeding is improved.

The invention provides 2,5-diketopiperazine dipeptide (I) derived from lysobacter enzymogenes as well as a preparation method and an application thereof and an endophytic bacterium producing 2,5-diketopiperazine dipeptide, namely lysobacter enzymogenes R-2-1. 2,5-diketopiperazine dipeptide (I) has the main beneficial effects that (1) lysobacter enzymogenes R-2-1 producing 2,5-diketopiperazine dipeptide (I) is provided and is the first lysobacter enzymogenes produced in the strain; (2) 2,5-diketopiperazine dipeptide (I) has strong inhabiting effects on aspergillus flavus and candida albicans, can serve as a novel bactericide and can be further used for preparing medicament for treating relevant diseases caused by aspergillus flavus or candida albicans; (3) 2,5-diketopiperazine dipeptide (I) is produced by liquid fermentation of lysobacter enzymogenes R-2-1 and has the advantages of simple and convenient operation process, short period, low cost and ensured origin; (4) 2,5-diketopiperazine dipeptide (I) is synthesized by a biological method and does not pollute the environment.

The invention belongs to the field of bio-genetic engineering and provides a shuttle plasmid and a method for efficiently expressing cecropin and lysozyme genes. The invention constructs an escherichia coli-bacillus subtilis shuttle plasmid suitable for being expressed in prokaryotes (escherichia coli, bacillus subtilis and bacillus sphaericus). Cecropins expressed in the prokaryotes does not have a toxic action on hosts. The shuttle plasmid can successfully express the fusion protein of various cecropins such as human lysozyme, bovine lactoferrin, and the like. 6His is introduced into the expression plasmid constructed by the invention, which is convenient to purify, and the purified fusion protein identifies and correctly cut enzyme cutting sites among all fusion peptide segments through lysostaphin so as to release all functional peptides and enable the functional peptides to recover the activity; and moreover, enzyme separation and cutting are also saved, thus the method can be effectively, simply and conveniently used. The shuttle plasmid also has a certain common availability, and all suitable genes can be cloned and expressed.

The present invention relates to type G lysozyme, and is especially type G lysozyme gene of and its coded protein and cloning method. The type G lysozyme gene of gulf scallop has the base sequence shown in SEQ ID No. 1, and its coded protein has the amino acid sequence shown in SEQ ID No 2. The type G lysozyme gene of gulf scallop the present invention clones has polyadenyl acid tailing signal and polyadenyl acid tail, has important function in immunologic defence of gulf scallop, and may be used in the recombinant expression and gene transfer of lysozyme. The present invention lays foundation for the disease prevention and treatment, gene aided selective breeding and medicine development of gulf scallop.

The invention relates to the use of lysozyme and restructured lysozyme 134 pure product or stock solution in preparing pharmaceutical preparations for the prevention and treatment for birds including chickens, ducks, geese, quails and livestock including pigs, sheep, cows, horses, donkeys, rabbits and honey bees. Wherein the restructured lysozyme 134 pure product or the combination of stock solution and antibiotics are employed to treat the diseases caused by viruses, Staphylococcus aureus, streptococcus, bacillus coli, salmonella, mycoplasma and Chlamydia.